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PubMed2005

Serological survey of Babesia bovis, Babesia bigemina, and Anaplasma marginale antibodies in cattle from the semi-arid region of the state of Bahia, Brazil, by enzyme-linked immunosorbent assays.

The objectives of this work were to determine the prevalence of Babesia bovis, Babesia bigemina, and Anaplasma marginale detecting antibodies in cattle raised in the semi-arid region of the state of Bahia, Brazil, through indirect enzyme linked immunosorbent assays (ELISA) and to compare the performances of indirect enzyme-linked immunosorbent assays with crude (I-ELISA-CrAnaAg) and recombinant major surface protein-5 (I-ELISA-MSP-5Ag), as antigens to detect antibodies against A. marginale. An stable enzootic area was found in Senhor do Bonfim and Euclides da Cunha for B. bovis that showed 86 and 95.5% of prevalence, respectively, and for B. bigemina with 90.8 and 91.3%. On the other hand, Uauá and Juazeiro, were characterized as enzootically unstable areas, since prevalences were: B. bovis--63.7 and 56.4% and B. bigemina--53 and 54.8%, respectively. The prevalence of A. marginale in the four municipalities was above 97% with I-ELISA-CrAnaAg and 94.8% with I-ELISA-MSP-5Ag which is an indication of stable enzootic condition for the rickettsia. The I-ELISA-CrAnaAg and I-ELISA-MSP-5Ag showed a highly significant association (r = 0.977), which means that both ELISA tests are suitable for epidemiological studies of A. marginale.

Anaplasma marginale↗
PubMed2000

Development of an ELISA system for detection of anti-Anaplasma marginale antibodies in cattle in Brazil.

An ELISA test was developed for detecting antibodies against Anaplasma marginale in bovine sera. Four antigenic preparations were produced from infected red blood cells. Some aliquots of this preparation were stored at -70 degrees C with 30% DMSO in phosphate-buffered saline (PBS) and others were lysed with 0.9% NH4Cl and stored at -20 degrees C. Typical anaplasmal structures were seen by electron microscopy in the antigenic preparations containing the erythrocytes that had been stored with DMSO. The performance of the ELISA test was evaluated by testing 298 positive serum samples collected from immunized cattle, 39 negative serum samples collected from cattle imported from areas free of A. marginale and 50 samples collected from cattle naturally infected in the field. The test gave a specificity of 94.87% and a sensitivity of 100%.

Anaplasma↗
PubMed2003

Anaplasma marginale msp1alpha genotypes evolved under positive selection pressure but are not markers for geographic isolates.

Anaplasma marginale (order Rickettsiales, family Anaplasmataceae), a tick-borne pathogen of cattle, is endemic in tropical and subtropical regions of the world. Many geographic isolates of A. marginale occur in the United States and have been identified by major surface protein 1a (MSP1a), which varies in sequence and molecular weight due to different numbers of tandem 28- to 29-amino-acid repeats. The present study was undertaken to examine the genetic variations among isolates of A. marginale obtained during 2001 from infected cattle from east-central Oklahoma, where A. marginale is endemic. The gene and protein sequences of MSP1a and msp4 nucleotide sequences were used to infer the phylogenetic relationships among Oklahoma and New World isolates from Argentina, Brazil, Mexico, and the United States. All 11 A. marginale isolates collected from Oklahoma had different MSP1a sequences but identical MSP4 sequences. The phylogenies of the msp4 sequences of 13 isolates from Oklahoma in comparison with those of 7 Latin American isolates and 12 U.S. isolates by maximum-parsimony (MP) and maximum-likelihood (ML) analyses, with A. centrale and A. ovis sequences used as outgroups, provided strong bootstrap analysis support for a Latin American clade. Isolates of A. marginale from the southern United States (Florida, Mississippi, and Virginia) and the west-central United States (California, Idaho, Illinois, Oregon, Missouri, and Texas) also grouped into two clades. Both clades contained isolates from Oklahoma, suggesting extensive cattle movement. ML analysis of the msp4 sequences of isolates from Oklahoma provided bootstrap analysis support for east-central and north-central clades in Oklahoma, and both clades included isolates from Stillwater, Okla. Analysis of the codon and amino acid changes among the msp4 sequences of isolates with different phylogenies provided evidence that msp4 is not under positive selection pressure. In contrast, the phylogenies of the MSP1a DNA and protein sequences of 13 isolates from Oklahoma in comparison with those of 7 Latin American and 13 isolates from the United States by MP and ML analyses demonstrated no geographic clustering and provided evidence that this gene is under positive selection pressure. The results indicate that msp1alpha is not a marker for the characterization of A. marginale geographic isolates and suggest that the genetic heterogeneity observed among isolates of A. marginale within Oklahoma could be explained by cattle movement and the maintenance of different genotypes by independent transmission events.

Amino Acid Sequence↗
PubMed1990

Intermediate site of development of Anaplasma marginale in feeding adult Dermacentor andersoni ticks that were infected as nymphs.

The development of Anaplasma marginale in midgut epithelial cells was studied in feeding, transmitting adult Dermacentor andersoni ticks. Laboratory-reared ticks experimentally infected as nymphs were allowed to feed from 1 to 9 days on susceptible calves. Gut tissues from ticks were collected on each day they fed (total, 9 days) and were processed for light and transmission electron microscopy. Colonies of A marginale were abundant during the first 6 days of feeding, after which numbers decreased. Colonies were adherent to the basement membrane of gut cells early during feeding, with resultant flattening of the colonies. Colonies also were seen in muscle cells on the hemocoel side of the basement membrane. Morphologic features of A marginale within muscle cells varied and were similar to those observed in gut cells. In addition, however, a large reticulated form in the colonies was observed in muscle cells and appeared to give rise to small particles by budding. Development of A marginale in muscle cells appears to represent an intermediate site of development between those in gut and in salivary glands.

Anaplasma↗
PubMed2004

Characterization of the Anaplasma marginale msp2 locus and its synteny with the omp1/p30 loci of Ehrlichia chaffeensis and E. canis.

Major surface protein 2 (MSP2) is an immunodominant and antigenically variant protein in the outer membrane of the rickettsia Anaplasma marginale. MSP2 variation is generated by recombination into a single operon-linked genomic expression site. The complete 5.6-kb msp2 locus was identified by sequencing a 90-kb region of the St. Maries strain of A. marginale. The locus encoded, in a 5' to 3' direction, a transcriptional regulator followed by five outer membrane proteins, OMP1, OpAG3, OpAG2, OpAG1, and MSP2. The sequences of this entire locus were analyzed using six genetically and phenotypically distinct strains of A. marginale. The overall locus structure was highly conserved with 100% identity among strains in the transcriptional regulator. Synonymous and nonsynonymous exchanges were infrequent in omp1 and rare in opag1 and opag2 among the six strains without strong bias for either type of exchange (neutral mutations). In contrast, mutations in opag3 seem to underlie purifying (negative) selection reflecting pressure to retain protein structure, in marked contrast to the highly antigenically variant MSP2. Interestingly, the 5' structure of this A. marginale msp2 locus is conserved in the omp1 gene locus of Ehrlichia chaffeensis and p30 gene locus of E. canis despite marked divergence between genera in the structure of the 3' region of the loci. This supports the hypothesis that the expression sites of these important immunogenic proteins are derived from a common precursor with later divergent evolution along genus lines.

Anaplasma marginale↗
PubMed2006

Comparison between indirect enzyme-linked immunosorbent assays for Anaplasma marginale antibodies with recombinant major surface protein 5 and initial body antigens.

Indirect enzyme-linked immunosorbent assays (ELISAs) based on recombinant major surface protein 5 (rMSP5) and initial body (IB) antigens from a Brazilian isolate of Anaplasma marginale were developed to detect antibodies against this rickettsia in cattle. Both tests showed the same sensitivity (98.2%) and specificities (100% for rMSP5 and 93.8% for IB ELISA) which did not differ statistically. No cross-reactions were detected with Babesia bigemina antibodies, but 5 (rMSP5 ELISA) to 15% (IB ELISA) of cross-reactions were detected with B. bovis antibodies. However, such difference was not statistically significant. Prevalences of seropositive crossbred beef cattle raised extensively in Miranda county, state of Mato Grosso do Sul, Brazil, were 78.1% by rMSP5 ELISA and 79.7% by IB ELISA. In the analysis of sera from dairy calves naturally-infected with A. marginale, the dynamics of antibody production was very similar between both tests, with maternal antibodies reaching the lowest levels at 15-30 days, followed by an increase in the mean optical densities in both ELISAs, suggesting the development of active immunity against A. marginale. Results showed that all calves were seropositive by one-year old, characterizing a situation of enzootic stability. The similar performances of the ELISAs suggest that both tests can be used in epidemiological surveys for detection of antibodies to A. marginale in cattle.

Anaplasma marginale↗
PubMed1987

Characterization of an immunoprotective protein complex of Anaplasma marginale by cloning and expression of the gene coding for polypeptide Am105L.

Immunization with an Anaplasma marginale surface protein complex containing two polypeptides (Am105U and Am105L), each having a molecular weight of 105,000, protected cattle against challenge with virulent organisms. These polypeptides were immunoprecipitated together from detergent extracts of A. marginale by a neutralizing monoclonal antibody. After surface radioiodination of intact parasites, both Am105U and Am105L contained the radiolabel. To define the structural and antigenic relationships between Am105U and Am105L and to determine individual efficacies as protective immunogens, we cloned and expressed A. marginale DNA in Escherichia coli. We identified recombinant bacteria which expressed a novel protein of 105,000 molecular weight as a major cellular component. The recombinant protein was structurally and antigenically homologous to Am105L. There were multiple, partially homologous copies of the cloned DNA sequence in the rickettsial genome.

Anaplasma↗
PubMed2003

Characterization of the functional domain of major surface protein 1a involved in adhesion of the rickettsia Anaplasma marginale to host cells.

The major surface protein (MSP) 1a of the genus type species Anaplasma marginale (Rickettsiales: Anaplasmataceae) has been shown to mediate adhesion, infection and transmission of the organism, as well as to contribute to protective immunity in cattle. MSP1a contains a variable number of tandemly repeated peptides in the amino-terminal region, while the remainder of the protein is highly conserved among isolates. The number of repeats varies among geographic isolates of A. marginale but is constant within an isolate and has been used as a stable genetic marker of isolate identity. Because the sequence of the tandem repeats is the most variable part of the protein among isolates, this region of the protein is most likely to be involved in adhesion to host cells, a prerequisite to infection. The purpose of this study was to characterize the organization and function of the MSP1a tandem repeats of A. marginale in adhesion to host cells. We demonstrated by use of recombinant mutant proteins that the tandemly repeated region of MSP1a was necessary and sufficient to mediate adhesion of MSP1a to tick cells and bovine erythrocytes. Synthetic peptides representing the predominant sequences of individual repeats were tested for their adhesive capacity for tick cell extract (TCE). Peptides containing acidic amino acids D or E at position 20 bound to TCE, while peptides with a G as the 20th amino acid were not adhesive to TCE. Antibodies produced in rabbits against a synthetic repeat peptide neutralized A. marginale infection of cultured tick cells, and the neutralization observed was similar to that effected by antibodies produced against the whole MSP1a recombinant protein. Analysis of tandemly repeated MSP1a peptides of several geographic isolates of A. marginale revealed a complex relationship between the msp1alpha genotype and the tick-transmissible phenotype of the isolate and suggested that both the sequence and conformation of the repeated peptides influenced the adhesive properties of MSP1a. These studies demonstrated that the tandemly repeated region of the protein mediates the adhesive function of MSP1a.

Amino Acid Sequence↗
PubMed2004

Anaplasma marginale major surface protein 2 CD4+-T-cell epitopes are evenly distributed in conserved and hypervariable regions (HVR), whereas linear B-cell epitopes are predominantly located in the HVR.

Organisms in the genus Anaplasma express an immunodominant major surface protein 2 (MSP2), composed of a central hypervariable region (HVR) flanked by highly conserved regions. Throughout Anaplasma marginale infection, recombination results in the sequential appearance of novel MSP2 variants and subsequent control of rickettsemia by the immune response, leading to persistent infection. To determine whether immune evasion and selection for variant organisms is associated with a predominant response against HVR epitopes, T-cell and linear B-cell epitopes were localized by measuring peripheral blood gamma interferon-secreting cells, proliferation, and antibody binding to 27 overlapping peptides spanning MSP2 in 16 cattle. Similar numbers of MSP2-specific CD4(+) T-cell epitopes eliciting responses of similar magnitude were found in conserved and hypervariable regions. T-cell epitope clusters recognized by the majority of animals were identified in the HVR (amino acids [aa] 171 to 229) and conserved regions (aa 101 to 170 and 272 to 361). In contrast, linear B-cell epitopes were concentrated in the HVR, residing within hydrophilic sequences. The pattern of recognition of epitope clusters by T cells and of HVR epitopes by B cells is consistent with the influence of protein structure on epitope recognition.

Anaplasma marginale↗
PubMed1993

Derivation of the complete msp4 gene sequence of Anaplasma marginale without cloning.

The gene encoding MSP4, a 31-kDa surface protein of the rickettsia Anaplasma marginale, was completely sequenced without prior cloning of the gene. Degenerate oligodeoxyribonucleotides (oligos) corresponding to the N-terminal amino-acid sequence of MSP4 were used as primers in the polymerase chain reaction (PCR) to amplify a 65-bp fragment of which the central 32 bp was nondegenerate. The 32-bp probe was hybridized to restriction enzyme-digested, genomic A. marginale DNA in Southern blots, and the hybridizing fragment was size selected from agarose gels and ligated into the Bluescript II SK (+/-) plasmid. The ligation reaction mixture was used as a template for PCR, amplified from the 32-bp oligo or its inverse complement to plasmid sequences. The PCR products were sequenced to obtain the entire msp4 gene and surrounding regions. This method of sequencing a gene without previously obtaining a clone for that gene could be beneficial in situations where conventional cloning and screening strategies are inefficient or ineffective.

Amino Acid Sequence↗
PubMed1994

Putative adhesins of Anaplasma marginale: major surface polypeptides 1a and 1b.

Genes for the MSP1a and MSP1b subunits of the Anaplasma marginale surface antigen complex MSP1 were previously cloned and expressed in Escherichia coli. We report here the localization of MSP1a and MSP1b polypeptides on the surface of recombinant E. coli by using a live cell indirect immunofluorescent antibody assay. Recombinant E. coli cells expressing the msp1 alpha gene or the msp1 beta gene encoding the MSP1a and MSP1b polypeptide subunits, respectively, were shown by a culture recovery adhesion assay and by direct microscopic examination to specifically adhere to bovine erythrocytes. This adhesion was more than additive when both genes were coexpressed in a single recombinant construct. Similarly, these recombinants hemagglutinated bovine erythrocytes in a microtiter hemagglutination assay. Inhibition of recombinant E. coli adhesion to bovine erythrocytes and hemagglutination inhibition were observed in the presence of homologous monospecific polyclonal antiserum raised against purified MSP1a or MSP1b polypeptide. These data suggest that the MSP1a and MSP1b polypeptides have functions as adhesins on A. marginale initial bodies, probably during erythrocyte invasion.

Anaplasma↗
PubMed2004

The effect of management factors on the seroprevalence of Anaplasma marginale in Bos indicus cattle in the Mexican tropics.

A cross-sectional study with a two-stage design and proportional distribution was carried out to determine the effect of management factors on the seroprevalence of Anaplasma marginale in Bos indicus cattle in the Mexican tropics. Serum was obtained from 384 cattle aged 1-2 years on 92 farms. The number of samples was proportionally distributed according to the number of farms in eastern Yucatan. Antibody activity against A. marginale was assessed using the card agglutination test. A primary screening using a 2 x K contingency table of the exposed variables with the outcomes was performed. All variables for which p < 0.20 were included in a fixed-effects log regression. The seroprevalence in the cattle was 69.75% (SE +/- 0.02). Sixty-four per cent of the farms had a seroprevalence > or = 75%. The risks related to managemental factors were stocking density ( > or = 1 animal/ha, OR = 10.94), type of acaricide (pyrethroids, OR = 3.8), dipping interval ( > 60 days, OR = 0.13) and type of veterinary instruments used (needles, scalpels, ear tattoos, and dehorners, OR = 0.17).

Agglutination Tests↗
PubMed1978

Efficacy of attenuated Anaplasma marginale vaccine under laboratory and field conditions in Colombia.

Four-month-old Holstein-Friesian calves were inoculated with 3 different doses (1, 2, and 3 ml) of attenuated Anaplasma marginale vaccine. Vaccinated calves showed mild anaplasma parasitemia, slight decrease in packed cell volume, low serologic conversion, and no clinical illness. An artificial challenge exposure of vaccinated and unvaccinated calves with virulent Colombian A marginale showed that the vaccine provided protection against clinical signs of the disease, including parasitemia and anemia. The volume of the vaccinal dose did not alter the degree of protection provided. A 2nd group of 8- to 9-month-old Holstein-Friesian calves was then inoculated with 3 ml of anaplasma vaccine and premunized with both Babesia bigemina and Babesia argentina while being housed in an area free of these diseases. Calves were moved to an enzootic region heavily infested with various arthropods, including ticks, for natural field challenge exposure. Control calves, which were not given anaplasma vaccine, suffered clinical illness manifested by severe anemia and an average weight loss of 50.6 kg due to anaplasma field challenge exposure. In contrast, vaccinated calves did not show anemia and their weight loss was 3.9 kg.

Anaplasma↗
PubMed2005

Detection of antibodies against Anaplasma marginale in milk using a recombinant MSP5 indirect ELISA.

An indirect enzyme linked immunosorbent assay (iELISA) for diagnosis of anaplasmosis using undiluted individual milk samples from dairy cows was developed. The recombinant 19 kDa major surface protein 5 (rMSP5) of Anaplasma marginale was used as antigen. A monoclonal antibody against bovine IgG1 conjugated with peroxidase and the chromogen 3,5,3',5'-tetramethylbenzidine were used in the test. Strong and weak, positive and negative milk samples were set up as reference controls. Results were expressed as percentage of positivity (PP) contrasting with the strongest positive control. The test was evaluated in two groups (G1 and G2) of lactating dairy cows from herds located in A. marginale non-endemic areas of Argentina. The infection status of both groups, G1 (n=128) sampled after anaplasmosis outbreak, and G2 (n=216) free of anaplasmosis was established by polymerase chain reaction (PCR). Serum samples of cows from G1 and G2 were analyzed by card agglutination test (CAT) and competitive ELISA (cELISA), while the novel iELISA was evaluated in their corresponding milk samples. At a cutoff of 42 PP, the ELISA has 98% sensitivity and 95% specificity. A significant difference (P<0.0001) was found between the mean PP value of negative samples from G1 (17.4+/-14.9), and G2 (8.6+/-7.1). The agreement and kappa (kappa) value between iELISA and PCR was 96%, kappa=0.919; between iELISA and CAT was 97%, kappa=0.880; and between iELISA and cELISA was 97%, kappa=0.899. These results strongly support the usefulness of iELISA to detect A. marginale antibodies in milk. Additional studies are necessary to define the ability of the milk iELISA to detect not only acutely infected, but also carrier cattle.

Agglutination Tests↗
PubMed1977

Cell-mediated immune response to virulent and attenuated Anaplasma marginale administered to cattle in live and inactivated forms.

The leukocyte migration-inhibition test (LMIT) and lymphocyte transformation of blood leukocytes from cattle exposed to virulent, attenuated, and inactivated preparations of Anaplasma marginale served as indices of cell-mediated immunity in bovine anaplasmosis. The LMIT response increased markedly in cattle vaccinated with attenuated A marginale or injected with virulent A marginale. Cattle inoculated with inactivated anaplasma developed a transient, low-level LMIT response. The response to antigenic stimulation recorded in the lymphocyte transformation test of leukocytes collected from cattle which were given live anaplasma was less than the response elicited from leukocytes collected from cattle vaccinated with inactivated anaplasma in adjuvant.

Anaplasma↗
PubMed2006

Prevalence of antibodies to serotypes of Bluetongue virus and Anaplasma marginale in Montana feeder cattle: 2002-2003.

In a serologic survey of Montana-source weaned calves and yearling cattle, the apparent prevalence of antibodies to Bluetongue virus was 0.68% and 1.26% in 2002 and 2003, respectively, and to Anaplasma marginale at a positive cutoff at 30% inhibition it was 1.82% and 1.35% in 2002 and 2003, and at a positive cutoff at 42% inhibition it was 0.76% and 0.55% in 2002 and 2003, respectively, suggesting that the risk of importing infected animals was very low.

Anaplasma marginale↗